l-Asparaginase from Proteus vulgaris

To produce an immunologically and enzymologically new type of l-asparaginase, 108 strains of bacteria were screened for enzyme production. As a result, 13 bacteria belonging to the genera Alcaligenes, Bacterium, and Proteus were found to produce l-asparaginases in high levels. Among these l-asparaginases, partially purified l-asparaginases from B. cadaveris and P. vulgaris showed antitumor activity. A partially purified l-asparaginase preparation of P. vulgaris did not react with the antibody of Escherichia colil-asparaginase on the Ouchterlony agar plate. Culture conditions for the production of l-asparaginase by P. vulgaris were investigated in detail. The enzyme was produced in high yields when cells were grown aerobically in a medium containing sodium fumarate and corn steep liquor. The addition of glucose or ammonium ion to the medium, however, resulted in depressed production of l-asparaginase. Under the optimum conditions, 3,700 international units of l-asparaginase was obtained from 1 liter of culture medium.     

L-asparaginase production by isolated Staphylococcus sp. - 6A: design of experiment considering interaction effect for process parameter optimization

AIMS: Evaluation of fermentation process parameter interactions for the production of l-asparaginase by isolated Staphylococcus sp. - 6A. METHODS AND RESULTS: Fractional factorial design of experimentation (L18 orthogonal array of Taguchi methodology) was adopted to optimize nutritional (carbon and nitrogen sources), physiological (incubation temperature, medium pH, aeration and agitation) and microbial (inoculum level) fermentation factors. The experimental results and software predicted enzyme production values were comparable. CONCLUSION: Incubation temperature, inoculum level and medium pH, among all fermentation factors, were major influential parameters at their individual level, and contributed to more than 60% of total l-asparaginase production. Interaction data of selected fermentation parameters could be classified as least and most significant at individual and interactive levels. Aeration and agitation were most significant at interactive level, but least significant at individual level, and showed maximum severity index and vice versa at enzyme production. SIGNIFICANCE AND IMPACT OF THE STUDY: All selected factors showed impact on l-asparaginase enzyme production by this isolated microbial strain either at the individual or interactive level. Incubation temperature, inoculum concentration, pH of the medium and nutritional source (glucose and ammonium chloride) had impact at individual level, while aeration, agitation and incubation time showed influence at interactive level. Significant improvement (ninefold increase) in enzyme production by this microbial isolate was noted under optimized environment.     

Optimization of BLyS production and purification from Escherichia coli

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.     

Optimization of a heterologous pathway for the production of flavonoids from glucose

The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides have been made recently in improving strain titers and yields, current fermentation strategies suffer from two major drawbacks-(1) the requirement for expensive phenylpropanoic precursors supplemented into the media and (2) the need for two separate media formulations for biomass/protein generation and flavonoid production. In this study, we detail the construction of a series of strains capable of bypassing both of these problems. A four-step heterologous pathway consisting of the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) was assembled within two engineered l-tyrosine Escherichia coli overproducers in order to enable the production of the main flavonoid precursor naringenin directly from glucose. During the course of this investigation, we discovered that extensive optimization of both enzyme sources and relative gene expression levels was required to achieve high quantities of both p-coumaric acid and naringenin accumulation. Once this metabolic balance was achieved, however, such strains were found to be capable of producing 29 mg/l naringenin from glucose and up to 84 mg/l naringenin with the addition of the fatty acid enzyme inhibitor, cerulenin. These results were obtained through cultivation of E. coli in a single minimal medium formulation without additional precursor supplementation, thus paving the way for the development of a simple and economical process for the microbial production of flavonoids directly from glucose.     

High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures

The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N-glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems.     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization

Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trx-pedA) expression approach. Trx-PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications.     

High-level expression and purification of biologically active recombinant pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) from the leaves of the pokeweed plant, Phytolacca americana, is a naturally occurring single-chain ribosome-inactivating protein, which catalytically inactivates both prokaryotic and eukaryotic ribosomes. The therapeutic potential of PAP has gained considerable interest in recent years due to the clinical use of native PAP as the active moiety of immunoconjugates against cancer and AIDS. The clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of a homogenously pure and active PAP preparation with minimal batch to batch variability from its natural source. Previous methods for expression of recombinant PAP in yeast, transgenic plants and Escherichia coli have resulted in either unacceptably low yields or were too toxic to the host system. Here, we report a successful strategy which allows high level expression of PAP as inclusion bodies in E. coli. Purification of refolded recombinant protein from solubilized inclusion bodies by size-exclusion chromatography yielded biologically active recombinant PAP (final yield: 10 to 12 mg/L). The ribosome depurinating in vitro N-glycosidase activity and cellular anti-HIV activity of recombinant PAP were comparable to those of the native PAP. This expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PAP sufficient to carry out advanced clinical trials. To our knowledge, this is the first large-scale expression and purification of biologically active recombinant PAP from E. coli.     

Purification and properties of L-asparaginase from Serratia marcescens

The purification and properties of a tumor inhibitory l-asparaginase from Serratia marcescens are described. The following properties of the enzyme were examined: kinetics of the enzyme reaction, catalytic activity as a function of pH, boundary sedimentation velocity, electrophoresis on polyacrylamide gel, immuno-electrophoresis against homologous and heterologous antisera, immunodiffusion, blood clearance rate in mice, and inhibition of the 6C3HED lymphoma in C3H mice. Complete regression of this tumor was obtained with a smaller dose of the enzyme from S. marcescens than with enzyme from Escherichia coli. The reason for this difference was not evident from a comparison of several properties of the two enzymes.     

Production of hydrolytic enzymes by oral isolates of Eikenella corrodens

Abstract Thermus thermophilus cells harboring an expression plasmid for the aqualysin I gene secrete the mature enzyme into the medium. In an Escherichia coli expression system, a precursor of the enzyme with the C-terminal pro-sequence is accumulated in the cells, and upon treatment at 65°C the active enzyme is produced. One- to 10-amino acid residue deletions, as well as complete 105-residue deletion of the C-terminal pro-sequence from the C-terminus, did not affect the production of the enzyme in T. coli cells. T. thermophilus cells harboring plasmids for mutant precursors with one- and three-residue deletions secreted the enzyme extracellularly. However, transformants harboring plasmids for mutant precursors with deletions of five or more amino acid residues could not be obtained. These results suggest that the C-terminal pro-sequence plays an important role in the extracellular secretion of the enzyme in T. thermophilus cells.     

Xylanase of the micromycete Rhizopus var. microsporus 595: preparation, structural and functional characteristics, and application

Procedures for the production of endo-1,4-beta-xylanase have been developed. An active producer-Rhizopus var. microsporus BKMF-595-has been chosen, and the conditions of surface and submerged cultivation, as well as the composition of the culture medium for this strain, have been optimized to ensure maximum yield of the target enzyme. Activity of xylomicrosporin Px equaled 123 U/g, while the activity of xylomicrosporin Gx equaled 25 U/cm3. Homogeneous enzyme preparations, purified 59.44-fold and 72.6-fold, have been obtained. The dependence of endo-1,4-beta-xylanase catalytic activity on temperature and pH of the reaction medium has been studied. The enzyme has been shown to be most stable in the pH range 5.0-6.0 and to be thermostable. Amino acid composition and subunit structure of the enzyme were determined; the molecular masses of the subunits equaled 50 and 56 kDa. Carboxyl groups of glutamic and aspartic acid residues of the active center were experimentally shown to play an important role in catalysis. The potential of this enzyme for beer production has been demonstrated.     

Systematic analysis of fusion and affinity tags using human aspartyl-tRNA synthetase expressed in E. coli

Fusion and affinity tags are popular tools for the expression of mammalian proteins in bacteria. To facilitate the selection of expression approaches, a systematic comparison was performed. We cloned, sequenced, and expressed in Escherichia coli ubiquitin- and SUMO-hDRS fusion proteins with biotin- or 6xHis-tags. The tagging of hDRS with ubiquitin or SUMO was necessary to express properly folded and biologically active enzyme. Similar enhancement of hDRS activity was obtained by fusion to ubiquitin or SUMO. Ubiquitin, SUMO, biotin, and hexahistidine tags did not appreciably interfere with hDRS activity. Fusion proteins were specifically cleaved without altering the N-terminal of hDRS. After cleavage hDRS remained soluble and active with a specific activity comparable to that of the fused protein. Similar activity was observed with biotin- and 6xHis-tagging of hDRS. Higher purity but significantly lower yields of hDRS were obtained using biotin-tagging. Overall we demonstrated ubiquitin and SUMO fusion proteins similarly enhanced the proper folding of hDRS expressed in E. coli. In comparison to previous expressions of hDRS as a GST fusion, ubiquitin, and SUMO fusions provided higher yields and easier purification and cleavage.     

Pseudomonas aeruginosa amidase: aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonas aeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of soluble amidase, relatively to other proteins, was obtained in the condition at 37°C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all the tested growth conditions. Addition of ethanol at 25°C in the culture medium improved amidase yield, which quantitatively aggregated in a biological active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37°C by FTIR analysis demonstrated a lower content of intermolecular interactions which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.     

Localization and production of novel l-asparaginase from Pectobacterium carotovorum MTCC 1428

Bacterial l-asparaginase has been widely used as therapeutic agent in the treatment of various lymphoblastic leukemia diseases. Studies on localization and production of novel glutaminase-free l-asparaginase were performed using Pectobacterium carotovorum MTCC 1428. The localization of l-asparaginase was carried out using cell fractionation techniques. The activity of l-asparaginase was found to be 85 and 77% in the cytoplasm of P. carotovorum MTCC 1428 grown on medium containing l-asparagine and combination of l-asparagine and glucose respectively. Among the tested carbon sources, l-asparagine or the combination of l-asparagine and glucose was found to be the most suitable carbon sources to maximize the production of l-asparaginase. The maximum production of l-asparaginase was observed to be 14.56 U/ml (26.92 U/mg of protein) at 4 and 2 g/l of l-asparagine and glucose respectively. Yeast extract, l-asparagine and peptone have shown significant effect on the production of l-asparaginase. P. carotovorum MTCC 1428 has assimilated l-asparagine as an essential carbon source for maximizing the production of l-asparaginase.     

A comparison of the process issues in expressing the same recombinant enzyme periplasmically in Escherichia coli and extracellularly in Streptomyces lividans.

The choice of a host for the production of a biological molecule will have a significant effect on isolation and purification procedures employed. This paper makes a comparison between the production of a single enzyme, a recombinant alpha-amylase, in Escherichia coli and Streptomyces lividans, on a small scale. It defines the differences in the cultivation and in the isolation stages and also describes the impact of the expression system on later downstream processing steps. At the cultivation stage, the specific productivity of the E. coli in units per gram per hour is four times that of the S. lividans while the total biomass yields are of the same order. The initial volume for downstream processing of S. lividans is six-fold larger and the total protein released into the extracellular medium is three times greater than E. coli, however, the recoverable yield from the E. coli is a fifth of that obtained from the S. lividans and requires three additional stages prior to chromatography. Even with these stages the final specific activity is 64% of the S. lividans. The results indicate the need to consider the whole process when making such comparisons.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Fatty acid structural requirements for leukotriene biosynthesis

Utilizing a variety of fatty acids, differing in chain length, degree and position of unsaturation, we investigated the substrate specificity for the enzymatic production of biologically active slow reacting substances (SRS) and of the other leukotrienes. A cell-free enzyme system obtained from RBL-1 cells was used in this study. The primary structural requirement observed for the conversion by this lipoxygenase enzyme system was a delta 5,8,11 unsaturation in a polyenoic fatty acid. Such fatty acids as 20:4 (5,8,11,14) 20:5 (5,8,11,14,17), 20:3 (5,8,11), 19:4 (5,8,11,14) and 18:4 (5,8,11,14) were readily converted to compounds that comigrated with 5-HETE and 5,12-DiHETE and to biologically active SRS. Chain length did not have an influence on the formatin of these hydroxyacids. Fatty acids with the initial unsaturation at delta 4, delta 6, delta 7, or delta 8 were a poor substrate for the leukotriene enzyme system. Therefore, this lipoxygenase pathway in leukocytes is quite different from the lipoxygenase in platelets which does not exhibit this specificity.     

Cloning and heterologous expression of a broad specificity aminotransferase of Leishmania mexicana promastigotes1

We have previously reported that Leishmania mexicana promastigotes possess a broad substrate specificity aminotransferase (BSAT), able to transaminate aspartate, aromatic amino acids, methionine and leucine. We have confirmed now this unusual substrate specificity by cloning its gene and expressing in Escherichia coli the recombinant active protein. The amino acid sequence of BSAT shares over 40% identity with other eukaryotic and prokaryotic aspartate aminotransferases, thus showing that the enzyme belongs to the subfamily Ialpha of aminotransferases, and has only 6% identity with the tyrosine aminotransferase from Trypanosoma cruzi, which has a similar substrate specificity. The production of recombinant active enzyme in good yields opens up the possibility of obtaining its 3D-structure, in order to investigate the structural basis of the broad substrate specificity.     

Production of a New Type of Acid Carboxypeptidase of Molds of the Aspergillus niger Group

The ability of 88 fungi, which had been obtained as high-potency strains for acid proteinase production, to produce a new type of acid carboxypeptidase (having on optimal pH of about 3 for hydrolysis of benzyloxycarbonyl-glutamyltyrosine) in surface koji culture was determined. Among the aspergilli, substantial amounts of this new acid carboxypeptidase were produced by Aspergillus saitoi, A. usamii, A. awamori, A. inuii, and A. niger. Maximum yields of acid carboxypeptidase per gram of substrate were obtained by submerged culture in a medium containing 0.9% defatted soybean and 0.6% wheat bran. However, the maximum enzyme concentration per milliliter was obtained with a medium containing 3% defatted soybean and 2% wheat bran. The terminal pH could be controlled by varying the concentrations of soybean oil meal and wheat bran. The maximum enzyme production was reached after 4 days or more at 30 C.